The present invention relates to medicines for activating antitumor immunity and for treating autoimmune diseases as well as to diagnosis of tumors or autoimmune diseases. In particular, the present invention relates to novel tumor antigen proteins, novel genes therefor, novel tumor antigen peptides, and the like.
It is known that the immune system, particularly T cells, plays an important role in vivo in tumor rejection. Indeed, infiltration of lymphocytes having cytotoxic effects on tumor cells has been observed in human tumor foci (Arch. Surg., 126:200-205, 1990), and cytotoxic T lymphocytes (CTLs) recognizing autologous tumor cells have been isolated from melanomas without great difficulties (e.g., Immunol. Today, 8:385, 1987; J. Immunol., 138:989, 1987; and Int. J. Cancer, 52:52-59, 1992). In addition, the results of clinical treatment of melanomas by T cell introduction also suggest the importance of T cells in tumor rejection (J. Natl. Cancer. Inst., 86:1159, 1994).
Although it has long been unknown about target molecules for CTLs attacking autologous tumor cells, the recent advance in immunology and molecular biology has gradually begun elucidating such target molecules. Specifically, it has been found that using T cell receptors (TCRs), CTL recognizes a complex consisting of tumor antigen peptide and major histocompatibility complex (MHC) class I antigen, and thereby attacks autologous tumor cells.
Tumor antigen peptides are generated from tumor antigen proteins. Thus, the proteins are intracellularly synthesized and then degraded in cytoplasm into the peptides by proteasome. On the other hand, MHC class I antigens formed at endoplasmic reticulum bind to the above tumor antigen peptides, and are transported via cis Golgi to trans Golgi, i.e., the mature side, and expressed on the cell surface (Rinsho-Menneki, 27(9):1034-1042, 1995).
As such a tumor antigen protein, T. Boon et al. identified a protein named MAGE from human melanoma cells for the first time in 1991 (Science, 254:1643-1647, 1991), and thereafter several additional tumor antigen proteins have been identified from melanoma cells.
As described in the review by T. Boon et al. (J. Exp. Med., 183, 725-729, 1996), tumor antigen proteins hitherto identified can be divided into the following four categories.
Tumor antigen proteins belonging to the first category are those proteins which are expressed only in testis among normal tissues, while they are expressed in melanoma, head and neck cancer, non-small cell lung cancer, bladder cancer and others, among tumor tissues. Among tumor antigen proteins in this category are the above-described MAGE and analogous proteins constituting a family of more than 12 members (J. Exp. Med., 178:489-495, 1993), as well as BAGE (Immunity, 2:167-175, 1995) and GAGE (J. Exp. Med., 182:689-698, 1995), all of which have been identified from melanoma cells.
Although some of such tumor antigen proteins in this category are highly expressed in melanoma, their expression is observed in only 10 to 30% of patients having a particular tumor other than melanoma, and therefore, they can not be applied widely to treatments or diagnoses of various tumors.
Tumor antigen proteins belonging to the second category are those proteins which are expressed only in melanocytes and retina among normal tissues, while the expression is observed only in melanomas among tumor tissues. Since these tissue-specific proteins are highly expressed in melanomas, they function as tumor antigen proteins specific for melanomas. Among tumor antigen proteins in this category are tyrosinase (J. Exp. Med., 178:489-495, 1993), MART-1 (Proc. Natl. Acad. Sci. USA, 91:3515, 1994), gp100 (J. Exp. Med., 179:1005-1009, 1994), and gp75 (J. Exp. Med., 181:799-804, 1995), genes for which have all been cloned from melanoma cells. Additionally and separately identified Melan-A (J. Exp. Med., 180:35, 1994) has proved to be the same molecule as MART-1.
However, the tumor antigen proteins in this category can not be used widely for treatments or diagnoses of various tumors, since they are not expressed in tumors other than melanoma.
Tumor antigen proteins belonging to the third category are those proteins which yield, through tumor-specific mutations, tumor antigen peptides newly recognized by CTL. Among tumor antigen proteins in this category are mutated CDK4 (Science, 269:1281-1284, 1995), xcex2-catenin (J. Exp. Med., 183:1185-1192, 1996), and MUM-1 (Proc. Natl. Acad. Sci. USA, 92:7976-7980, 1995). In CDK4 and xcex2-catenin, a single amino acid mutation increases the binding affinity of the peptides to MHC class I antigen, and allows them to be recognized by T cells. In MUM-1, its intron normally untranslated is translated due to mutation, and the peptide thus generated is recognized by T cells. However, since such mutations occur at low frequency, they can not be applied widely to treatments or diagnoses of various tumors.
As a tumor antigen protein belonging to the fourth category, P15 has been identified from melanoma cells, which is a protein widely expressed in normal tissues and which is also recognized by CTL (J. Immunol. 154:5944-5955, 1995).
Tumor antigen proteins or peptides hitherto known have been identified as follows.
In such identification, a set of tumor cells and CTLs attacking the tumor cells (usually established from lymphocytes of the same patient from whom the tumor cells are obtained) are firstly provided. Then, the cells from this set are used to directly identify tumor antigen peptides, or used to determine the gene encoding the tumor antigen protein from which corresponding tumor antigen peptides are identified.
Specifically, in the case where tumor antigen peptides are directly identified, tumor antigen peptides bound to MHC class I antigens in tumor cells are extracted under acidic conditions, and separated into various peptides using high-performance liquid chromatography. Cells expressing MHC class I antigen, but not expressing tumor antigen protein (for example, B cells from the same patient), are then pulsed with such various peptides, and examined for their reactivity with CTL to identify tumor antigen peptides. Then, the sequences of the peptides thus identified are further determined by, for example, mass spectrometry. In this way, tumor antigen peptides derived from Pmel 17 which is the same molecule as gp100 have been identified from melanoma cells (Science, 264:716-719, 1994).
In order to firstly determine the gene encoding tumor antigen protein and then to identify therefrom corresponding tumor antigen peptides, the gene encoding tumor antigen protein may be cloned using molecular biological techniques. cDNAs are prepared from tumor cells, and cotransfected with MHC class I antigen gene into cells not expressing tumor antigen proteins (for example, COS cells), in order to express them transiently. The products thus expressed are then repeatedly screened on the basis of their reactivity with CTL, until the gene encoding tumor antigen protein may finally be isolated. In this way, the genes for the above-mentioned MAGE, tyrosinase, MART-1, gp100, and gp75 have been cloned.
In order to deduce and identify the presented tumor antigen peptides actually bound to MHC class I antigens on the basis of the information about such tumor antigen gene, the methods as described below are used. Firstly, fragments of the gene encoding tumor antigen protein, having various sizes, are prepared using, for example, PCR, exonucleases, or restriction enzymes, and cotransfected with MHC class I antigen gene into cells not expressing tumor antigen proteins (e.g., COS cells), in order to express them transiently. The region(s) which include tumor antigen peptides are then identified on the basis of their reactivity with CTL. Subsequently, peptides are synthesized. Cells expressing MHC class I antigen but not expressing tumor antigen proteins are then pulsed with the synthesized peptides, and examined for their reactions with CTL to identify the tumor antigen peptides (J. Exp. Med., 176:1453, 1992; J. Exp. Med., 179:24, 759, 1994). The sequence regularities (motifs) for peptides, which are bound and presented by certain types of MHC such as HLA-A1, -A0201, -A0205, -A11, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, and -Cw0602 have been known (Immunogenetics, 41:178-228, 1995), and therefore, candidates for tumor antigen peptides may also be designed by making reference to such motifs, and such candidate peptides may be practically synthesized and examined in the same way as described above (Eur. J. Immunol., 24:759, 1994; J. Exp. Med., 180:347, 1994).
Furthermore, it is another possibility that tumor antigen proteins expressed at high level in tumors are expressed also in normal tissues, and cause autoimmune diseases by inducing excessive immune response against such tumor antigen proteins. For example, it was reported that when a combination of a chemotherapeutic agent and IL-2 was used for treating melanomas, appearance of leukoderma was observed (J. Clin. Oncol., 10:1338-1343, 1992). This is probably because CTLs or antibodies against the complexes consisting of fragments of the tumor antigen protein expressed in melanomas (referred to as peptide fragments) and MHC class I antigens were inductively produced, and they affected normal skin tissues to develop leukoderma, an autoimmune disease-like symptom.
As described above, some of the known tumor antigen proteins are expressed only in limited tumors, and others are expressed only in a small number of patients having a particular tumor even if they are expressed in various kinds of tumor, and therefore, they can not be used widely for treatments or diagnoses of various tumors.
Thus, the present invention aims to provide tumor antigen proteins or fragments thereof (hereinafter referred to as xe2x80x9cpeptide fragmentsxe2x80x9d or as xe2x80x9ctumor antigen peptidesxe2x80x9d) which, unlike the known tumor antigen proteins or their peptide fragments, can be used for treatments or diagnoses of a wide variety of tumors including squamous cell carcinoma, or which can be applied to major part of patients having a particular tumor even if they can be used only for limited tumors, or which can be applied to various tumors as a therapeutic or diagnostic assistant in the treatment or diagnosis for such tumors.
Squamous cell carcinoma is one of the most common cancers in human. In particular, squamous cell carcinomas in esophageal cancer and lung cancer are known to be relatively resistant to current chemotherapy and radiotherapy. Also in this regard, it is desired to develop specific immunotherapies such as those which use tumor antigen proteins or corresponding tumor antigen peptides.
Furthermore, when one develops autoimmune disease due to excessively induced specific immunity raised by tumor antigen protein, it would be desirous treatments to specifically block such immune response using, for example, antisense DNA/RNA for the gene encoding tumor antigen proteins or antagonists for the tumor antigen peptides.
With the aim of obtaining tumor antigen protein or corresponding tumor antigen peptides which can be applied widely to treatments or diagnoses of various tumors including, in particular, squamous cell carcinoma, the present inventors tried to identify tumor antigen proteins from tumors other than melanoma.
Specifically, the present inventors established a squamous cell carcinoma cell line KE-4 derived from esophageal cancer (hereinafter referred to as esophageal cancer cell line KE-4 or simply as KE-4), and also established CTL (hereinafter referred to as KE-4CTL) which recognizes tumor antigen peptides restricted to HLA-A2601 which is a MHC class I antigen expressed in said KE-4 (Cancer Res., 55:4248-4253, 1995).
Fibroblast cell line VA-13 was then cotransfected with a recombinant plasmid of cDNA library prepared from KE-4 and a recombinant plasmid containing HLA-A2601 cDNA. The resulting transfectants were treated with KE-4CTL, and screened by measuring the amount of produced IFN-xcex3 to determine whether KE-4-CTL was activated. As a result, the inventors succeeded in cloning a novel gene encoding tumor antigen protein of the present invention for the first time from tumor cells other than melanoma.
Thus, the gist of the present invention relates to:
(1) DNA encoding a protein having the amino acid sequence shown in SEQ ID NO: 2(Identification Method: P) or a variant protein thereof in which one or more amino acid residues are substituted, deleted or added, said protein and variant protein thereof being capable of yielding, through its intracellular decomposition, peptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding state;
(2) DNA which comprises the base sequence shown in SEQ ID NO: 1(Identification Method E), or a variant DNA which hybridizes to said DNA under stringent conditions, the protein produced by expression of said DNA and variant DNA being capable of yielding, through its intracellular decomposition, peptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding state;
(3) medicines comprising DNA of the above item (1) or (2) as an active ingredient;
(4) expression plasmids comprising DNA of the above item (1) or (2);
(5) transformants transformed with the expression plasmid of the above item (4);
(6) tumor antigen proteins produced by expression of DNA of the above item (1) or (2);
(7) tumor antigen peptides comprising part of the protein of the above item (6) which can bind to MHC class I antigen to be recognized by T cells, or derivatives thereof having functionally equivalent properties;
(8) tumor antigen peptides of the above item (7) which comprise all or part of the amino acid sequence of positions 749-757, 736-744, 785-793, or 690-698 in the amino acid sequence of SEQ ID NO: 2, or derivatives thereof having functionally equivalent properties;
(9) medicines comprising, as an active ingredient, tumor antigen protein of the above item (6), tumor antigen peptide or derivative thereof defined in the above item (7) or (8). (10) antibodies which specifically bind to the tumor antigen proteins of the above item (6) or tumor antigen peptides of the above item (7) or (8); and (11) DNA comprising 8 or more bases having a sequence complementary to the coding or 5xe2x80x2 non-coding sequence of DNA having the base sequence shown in SEQ ID NO: 2 or RNA corresponding to said DNA, or chemically modified variant thereof.
DNAs of the present invention encode a novel tumor antigen protein, and may include a DNA which encodes a protein having the amino acid sequence shown in SEQ ID NO: 2 or a variant protein thereof in which one or more amino acid residues are substituted, deleted or added, said protein and variant protein being capable of yielding, through its intracellular decomposition, peptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding state, as well as DNA which comprises the base sequence shown in SEQ ID NO: 1 or variant DNA thereof which hybridizes to said DNA under stringent conditions, the protein produced by expression of said DNA and variant DNA being capable of yielding, through its intracellular decomposition, peptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding state.
As used herein, the phrase xe2x80x9cvariant protein thereof in which one or more amino acid residues are substituted, deleted, or addedxe2x80x9d refers to so-called variant proteins artificially prepared, to naturally-occurring polymorphism, or to proteins produced by mutation or modification but having functionally equivalent properties. DNAs encoding such variant proteins may be prepared using, for example, the methods described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd ed., vols. 1∫3 (Cold Spring Harbor Laboratory Press, New York, 1989), such as site-directed mutagenesis or PCR method. In this context, the number of amino acid residues to be substituted, deleted, or added should be such a number that permits the substitution, deletion or addition by well-known methods such as site-directed mutagenesis described above.
xe2x80x9cVariant DNA which hybridizes to DNA under stringent conditionsxe2x80x9d as described herein may be obtained using, for example, the methods described in Molecular Cloning mentioned above. In this context, xe2x80x9cstringent conditionsxe2x80x9d refers to, for example, such conditions that hybridization is conducted at 42xc2x0 C. in a solution containing 6xc3x97SSC (20 xc3x97SSC means 333 mM sodium citrate and 333 mM NaCl), 0.5% SDS, and 50% formamide, followed by washing in a solution of 0.1 xc3x97SSC and 0.5% SDS at 68xc2x0 C., or those conditions described in Nakayama el al., Bio-Jikken-Illustrated, vol. 2, xe2x80x9cIdenshi-Kaiseki-no-Kiso (Basis for Gene Analysis)xe2x80x9d, pp. 148-151, Shujunsha,.1995. For the purpose of this invention, the protein produced by expression of such hybridizable DNA should comprise a peptide segment which is capable of binding to MHC class I antigen and recognized by T cells.
As used herein, xe2x80x9cprotein and variant protein which are capable of yielding, through its intracellular decomposition, peptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding statexe2x80x9d (hereinafter, such protein is sometimes referred to as tumor antigen protein) means that partial peptide consisting of part of the amino acid sequence of such protein or variant protein can bind to MHC class I antigen, and that when bound to MHC class I antigen and presented on cell surface, the complex of the peptide fragment and MHC class I antigen can be recognized by T cells capable of specifically binding thereto, and transduces signals to T cells. In this context, such binding means non-covalent binding.
In order to confirm that a given peptide fragment is capable of binding to MHC class I antigen and recognized by T cells, the peptide fragment may be bound to MHC class I antigen and presented on cell surface by expressing it endogenously in an appropriate cell or by adding it exogenously to an appropriate cell (pulsing). The peptide presenting cells may be then treated with T cells specific to the tumor antigen protein, and cytokines produced by the T cells may be measured. Alternatively, as a method measuring the cytotoxic activity of T cells against the peptide-presenting cells, a method using the peptidepresenting cells labeled with 51Cr (Int. J. Cancer, 58:317 (1994)) may also be used. In such methods, CTLs are preferably used as the T cells recognizing the peptide.
DNA of the present invention may be used as an active ingredient of medicines. For example, medicines which comprise DNA of the present invention as an active ingredient can be used for treating or preventing tumors by administering the DNA of the present invention to tumor patients. When DNA of the present invention is administered, the tumor antigen protein is expressed at high level in the cells. As a result, the tumor antigen peptides bind to MHC class I antigen and presented on the cell surface at high density. This will cause efficient proliferation of tumor-specific CTLs in the body, allowing treatment or prevention of the tumor. Administration and introduction of DNA of the present invention into cells may be achieved using viral vectors or according to any one of other procedures (Nikkei-Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36(1), 23-48 (1994); Jikken-Igaku-Zokan, 12(15), 1994, and references cited therein).
Examples of the methods using viral vectors include those methods in which DNA of the present invention is incorporated into DNA or RNA virus such as retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, poliovirus, or Sindbis virus, and introduced into cells. Among them, the methods using retrovirus, adenovirus, adeno-associated virus, or vaccinia virus are particularly preferred.
Other methods may include those in which expression plasmids are directly injected intramuscularly (DNA vaccination), the liposome method, Lipofectin method, microinjection, the calcium phosphate method, and electroporation, with DNA vaccination and the liposome method being particularly preferred.
In order to make DNA of the present invention act as medicine in practice, one can use either of two methods: in vivo method in which DNA is directly introduced into the body, or ex vivo method in which certain cells are removed from human, and after introducing DNA into said cells extracorporeally, reintroduced into the body (Nikkei-Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36(1), 23-48 (1994); Jikkenn-Igaku-Zokan, 12(15), 1994; and references cited therein). In vivo method is rather preferred.
In the case of in vivo methods, DNA may be administered by any appropriate route depending on the diseases and symptoms to be treated, and other factors. For example, it may be administered by intravenous, intraarterial, subcutaneous, intracutaneous, or intramuscular routes. In the case of in vivo methods, such medicines may be administered in various dosage forms such as solution, and they are typically formulated into injections containing DNA of the present invention as an active ingredient, which may also include, if necessary, conventional carriers. When DNA of the present invention is included in liposomes or membrane-fused liposomes (such as Sendai virus (HVJ)-liposomes), such medicines may be in the form of suspension, frozen drug, centrifugally-concentrated frozen drug or the like.
Although the amount of DNA of the present invention in such formulations may vary depending on, for example, the disease to be treated, the age and body weight of a particular patient, it is usually preferred to administer 0.0001-100 mg, more preferably 0.001-10 mg, of DNA of the present invention every several days to every several months.
Furthermore, the tumor antigen protein can be prepared in large quantities by recombinant DNA techniques using DNA of the present invention.
Preparation of tumor antigen protein by expression of DNA of the present invention may be achieved according to many publications and references such as Molecular Cloning mentioned above. An expression plasmid which can replicate and function in host cells is constructed by adding regulatory gene(s) such as a promoter which controlls transcription (e.g., trp, lac, T7, or SV40 early promoter) upstream to the DNA to be expressed and by inserting the resultant DNA into an appropriate vector (e.g., pSV-SPORT1). The expression plasmid is then introduced into appropriate host cells to obtain transformants. Examples of host cell include, for example, prokaryotes such as Escherichia coli, unicellular eukaryotes such as yeast, and cells derived from multicellular eukaryotes such as insects or animals. Gene transfer into host cells may be achieved by, for example, the calcium phosphate method, DEAE-dextran method, or the electric pulse method. Transformants cultured in appropriate medium produce the protein of interest. The tumor antigen protein thus obtained may be isolated and purified according to standard biochemical procedures.
In the present invention, xe2x80x9cpeptide fragments which can bind to MHC class I antigen and which can be recognized by T cells in such binding statexe2x80x9d, which may be produced through intracellular decomposition of tumor antigen protein of the present invention, i.e., xe2x80x9ctumor antigen peptidesxe2x80x9d, may be determined as follows.
Firstly, fragments of DNA encoding tumor antigen protein and having various sizes are prepared using, for example, PCR, exonucleases, or restriction enzymes, and then inserted into expression vectors as described above. The vectors are then cotransfected into cells not expressing tumor antigen proteins (e.g., COS cells), with a plasmid which comprises a gene for MHC class I antigen that presents tumor antigens, in order to express them transiently. The regions which include the tumor antigen peptides are identified on the basis of the reactivity of the transfectants with CTL. Subsequently, various peptides included in such regions are synthesized. Cells expressing MHC class I antigen which presents tumor antigens but not expressing tumor antigen proteins are pulsed with the synthesized peptides, and examined for their reaction with CTL to identify the tumor antigen peptides (J. Exp. Med., 176:1453, 1992; J. Exp. Med., 179:24, 759, 1994).
Alternatively, the sequence regularities (motifs) of antigen peptides bound and presented by certain MHC types such as HLA-A1, A0201, -A0205, -A11, -A24, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, and -Cw0602 have been known, and threfore, candidates for tumor antigen peptides may also be selected making reference to such motifs, and such candidate peptides may be synthesized and identified in the manner as described above (Eur. J. Immunol., 24:759, 1994; J. Exp. Med., 180:347, 1994).
It is also known that MHC includes class II antigens besides class I antigens, and that conjugates of such MHC class II antigen with particular tumor antigen peptides, which may be produced from tumor antigen protein through phagocytosis and decomposition by antigen-presenting cells, such as macrophage, will activate tumor-specific helper T cells (J. Immunol., 146:1708-1714, 1991).
The successful cloning of the novel tumor antigen protein gene of the present invention also enables those skilled in the art to determine additional tumor antigen peptides which bind to MHC class II antigen described above. Specifically, such antigen peptides may be determined on the basis of their reactivity with T cells or based on known information on motifs of such antigen peptides, in the same manner as MHC class I antigen.
The tumor antigen peptides thus determined may be prepared by usual methods known in peptide chemistry such as those described in xe2x80x9cPeptide Synthesisxe2x80x9d (Interscience, New York, 1966), xe2x80x9cThe Proteinsxe2x80x9d (vol. 2, Academic Press Inc., New York, 1976), xe2x80x9cPepuchido-Goseixe2x80x9d (Maruzen, 1975), or xe2x80x9cPepuchido-Gosei-no-Kiso-to-Jikkennxe2x80x9d (Maruzen, 1985). In particular, such peptide can be synthesized by selecting either the liquid phase method or the solid phase method depending on the structure of its C-terminus, with the liquid phase method being more preferable. Thus, peptides may be prepared by protecting and deprotecting functional groups in amino acids, and elongating them by a single residue or several residues. Protecting groups for functional groups on amino acids are described, for example, in the abovementioned publications concerning peptide chemistry.
For the purpose of the present invention, xe2x80x9ctumor antigen peptidesxe2x80x9d may be defined as peptide fragments derived from either a protein having the amino acid sequence shown in SEQ ID NO: 2 or a variant protein thereof as defined above. Although the following description mainly relates to tumor antigen peptides derived from the protein having the amino acid sequence shown in SEQ ID NO: 2 as well as derivatives thereof, it will be understood that such description can apply to tumor antigen peptides derived from variant proteins.
Tumor antigen peptides produced by intracellular decomposition of the protein shown in SEQ ID NO: 2 are not specifically restricted, and may include, but not limited to, those peptides that comprise all or part of the amino acid sequence of positions 749-757, 736-744, 785-793, or 690-698 in the amino acid sequence shown in SEQ ID NO: 2. Preferred are those peptides that consist of 9 amino acid residues, and those peptides that consists of the amino acid sequence of positions 749-757, 736-744, 785-793, or 690-698 in SEQ ID NO: 2 are particularly preferred. Regarding tumor antigen peptides described herein, for example, the peptide consisting of the amino acid sequence of positions 749-757 in SEQ ID NO: 2 is sometimes abbreviated as xe2x80x9c749-757xe2x80x9d.
As used herein, xe2x80x9cderivatives of tumor antigen peptidexe2x80x9d refers to those derivatives which have properties functionally equivalent to such tumor antigen peptide and in which some of the amino acid residues in said peptide are substituted, deleted, or added, or to those derivatives in which amino group(s) or carboxy group(s) in said peptide(s) or derivatives described just above are modified. In particular, examples of such derivatives may include those derivatives in which, in a tumor antigen peptide of the present invention comprising all or part of the amino acid sequence of positions 749-757, 736-744, 785-793, or 690-698 in the amino acid sequence of SEQ ID NO: 2, some of the amino acid residues in the amino acid sequence of positions 749-757, 736-744, 785-793, or 690-698 are substituted or deleted, or other amino acid residue(s) are added thereto.
Among derivatives in which some of the amino acid residues in said peptide are substituted, deleted, or added, preferred are those derivatives which retain the epitope regions in the tumor antigen peptides involved in their binding with CTL and in which amino acid residue(s) in the tumor antigen peptides involved in their binding with MHC class I antigen are substituted, deleted, or added. Among such derivatives, those derivatives in which a single amino acid residue is substituted are more preferred (Immunol. 84:298-303, 1995). For antigen peptides derived from melanoma tumor antigen protein gp 100, it is reported that substitution of amino acid(s) in the binding site for MHC class I antigen has resulted in its stronger binding with MHC class I antigen, and also caused stronger induction of CTL specific to such antigen peptide when used in in vitro stimulation of peripheral blood lymphocytes derived from melanoma patients (J. Immunol., 157:2539-2548, 1996).
Such derivatives can be easily synthesized using a commercially available peptide synthesizer, and the binding affinity of synthesized derivatives to MHC class I antigen may be easily measured by competitive inhibition assay between said derivatives and radiolabeled standard peptide for binding to MHC class I antigen (R. T. Kubo et al., J. Immunol., 152:3913, 1994). Thus, by subjecting various peptide derivatives to such assay, peptide derivatives having CTL-inducing activity can be easily selected. Since the peptide derivatives thus selected can bind to MHC class I antigen more strongly while retaining their binding ability to CTL, they can be used as more efficient tumor antigen peptides.
Examples of modifying group for amino group may include acyl groups, and in particular, alkanoyl groups of 1-6 carbon atoms, alkanoyl groups of 1-6 carbon atoms substituted by phenyl group, carbonyl groups substituted by cycloalkyl group of 5-7 carbon atoms, alkylsulfonyl groups of 1-6 carbon atoms, phenylsulfonyl groups, and the like.
Modifying group for carboxy group include, for example, ester and amide groups. Specific examples of such ester group may be alkyl ester groups of 1-6 carbon atoms, alkyl ester groups of 0-6 carbon atoms substituted by phenyl group, and cycloalkyl ester groups of 5-7 carbon atoms, and specific examples of such amide group may be an amide group, amide groups substituted by one or two alkyl groups of 1-6 carbon atoms, amide groups of 0-6 carbon atoms substituted by one or two alkyl groups substituted by phenyl, and amide groups forming a 5-7 membered azacycloalkane including the amide nitrogen as a ring member. xe2x80x9cAntibodiesxe2x80x9d of the present invention may be easily prepared according to, for example, the methods described in Lane, H.D. et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Labortory Press, New York, 1989). Specifically, antibodies which recognize tumor antigen proteins or tumor antigen peptides, and antibodies which further neutralize their activities may be easily prepared by immunizing an animal with such tumor antigen protein or tumor antigen peptide using conventional procedures. Such antibodies may be used in, for example, affinity chromatography, screening of cDNA library, immunological diagnosis, or preparation of medicines. Such immunological diagnosis may include immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent or luminescent assay, and the like.
As used herein, xe2x80x9cDNA comprising 8 or more bases having a sequence complementary to the coding sequence or 5xe2x80x2 non-coding sequence of DNA comprising the base sequence shown in SEQ ID NO: 1 or RNA corresponding to said DNAxe2x80x9d means an antisense strand of double stranded DNA, or RNA corresponding to such antisense strand DNA, comprising 8 or more bases (hereinafter referred to as antisense oligonucleotides).
For example, as such antisense oligonucleotides, DNA may be prepared on the basis of the base sequence of the gene encoding tumor antigen protein of the present invention, and corresponding RNA may be prepared by incorporating such DNA into an expression plasmid in the antisense direction.
Although such antisense oligonucleotides may have a sequence complimentary to any part of the coding sequence or 5xe2x80x2 non-coding sequence of DNA of the present invention comprising the base sequence shown in SEQ ID NO: 1, they preferably have a sequence complimentary to transcription initiation site, translation initiation site, 5xe2x80x2 non-translated region, a boundary region between exon and intron, or 5xe2x80x2 CAP region.
In the above description, xe2x80x9cchemically modified variantsxe2x80x9d of xe2x80x9cDNA or RNA corresponding to said DNAxe2x80x9d (hereinafter referred to as chemically modified variant of antisense oligonucleotides) may include those variants which have increased transferability into cells or increased stability in cells. Specific examples of the variants include phosphorothioate, phosphorodithioate, alkyl phosphotriester, alkyl phosphonate, or alkyl phosphoamidate derivatives (xe2x80x9cAntisense RNA and DNAxe2x80x9d, WILLEY-LISS, 1992, pp. 1-50). Such chemically modified variant may be prepared according to, for example, the above-mentioned reference.
Such antisense oligonucleotides or chemically modified variants thereof may be used to control expression of the gene encoding tumor antigen protein. Since such control can decrease the amount of tumor antigen protein to be produced, and thereby decrease a damage caused by CTLs and also inhibit proliferation of CTL, autoimmune diseases due to over-expression of tumor antigen protein may be treated or prevented by such approach.
When the antisense oligonucleotides or chemically modified variants thereof are administered as such, preferred length thereof may be 8-200 bases, more preferably 10-25 bases, and most preferably 12-25 bases.
When inserted into expression plasmids, preferred length of the antisense oligonucleotides may be 100 bases or more, preferably 300-1000 bases, and more preferably 500-1000 bases.
Antisense oligonucleotides inserted in expression plasmids may be introduced into cells according to, for example, the methods described in Jikken-Igaku, vol. 12 (1994), such as those employing liposomes or recombinant viruses. Expression plasmids for antisense oligonucleotides may be easily prepared using conventional expression vectors just by placing the genes of the present invention after the promoter in the opposite direction so that the genes of the present invention may be transcribed in the direction from 3xe2x80x2 to 5xe2x80x2.
When administered as such, antisense oligonucleotides or chemical variants of the antisense oligonucleotides may be formulated by mixing them with stabilizing agents, buffers, solvents, and/or the like, and then administered simultaneously with antibiotics, anti-inflammatory agents, or anesthetics. The formulations thus prepared may be administered via various routes. Such formulations are preferably administered everyday or every several days to every several weeks. Furthermore, in order to avoid such frequent administration, sustained-release minipellet formulation may also be prepared and implanted near the affected area. Alternatively, the formulation may be slowly administered in continuous manner using, for example, an osmotic pump. Dosage are typically to be adjusted so that the concentration at the site of action will be from 0.1 nM to 10 xcexcM.
Tumor antigen proteins, tumor antigen peptides, and derivatives thereof having functionally equivalent properties, of the present invention may be used alone or in combination, and medicines comprising them as an active ingredient may be administered together with adjuvants or in particulate dosage form in order to effectively establish the cellular immunity. Specifically, when tumor antigen protein or tumor antigen peptide is administered to a subject, tumor antigen peptides are presented at high density on MHC class I antigens of the antigen-presenting cells, resulting in efficient proliferation of tumor-specific CTLs. For such purpose, those adjuvants described in the literature (Clin. Microbiol. Rev., 7:277-289, 1994) are applicable. The active ingredient(s) are administered in a dosage form which allows the foreign antigen peptide to be efficiently presented on MHC class I antigen, such as liposomal preparations, particulate preparations in which the active ingredient(s) are bound to beads having a diameter of several xcexcm, or preparations in which the active ingredient(s) are bound to lipids. It may be also possible to administer antigen-presenting cells such as dendritic cells or macrophages pulsed with the tumor antigen peptide, or cells transfected with DNA encoding the tumor antigen protein. Although the dose of the tumor antigen protein or tumor antigen peptide of the present invention in such preparations may be appropriately adjusted depending on various factors such as the disease to be treated, age and body weight of a particular patient, preferred dose is between 0.0001 mg and 1000 mg, and more preferably between 0.001 mg and 1000 mg. It is preferably administered every several days to every several months.
A method for in vitro induction of CTL from peripheral lymphocytes using tumor antigen peptide of the present invention is exemplified as follows.
Peripheral blood lymphocytes from an esophageal cancer patient with squamous cell carcinoma are in vitro-cultured, and a tumor antigen peptide of the present invention, for example, a peptide having the sequence of xe2x80x9c736-744xe2x80x9d, xe2x80x9c749-757xe2x80x9d, xe2x80x9c785-793xe2x80x9d, or xe2x80x9c690-698xe2x80x9d is added to the culture medium at the final concentration of 10 xcexcg/ml, in order to stimulate the peripheral blood lymphocytes. Such stimulation is repeated three times at intervals of one week. One week after the third stimulation, the peripheral blood lymphocytes are recovered, and measured for their cytotoxic activity according to the methods described in D. D. Kharkevitch et al, Int. J. Cancer, 58:317 (1994), in order to find CTL-inducing activity of the tumor antigen peptide of the present invention.
The method of the present invention for diagnosing tumors or autoimmune diseases may be conducted using antibodies specifically binding to a tumor antigen protein or tumor antigen peptide. Examples of such method may include those detecting tumor antigen protein in tumor tissue preparations, or detecting the presence of tumor antigen protein or antibodies against tumor antigen protein in blood or tissues. Such detection may be achieved by any appropriate method selected from, for example, immunohistochemical methods, immunoblotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), fluorescent and luminescent assays. Furthermore, detection of tumor antigen protein using antibodies enables early detection of tumors or their recurrence, as well as selection of patients who may be suitably treated with the tumor antigen proteins, tumor antigen peptides, or DNA encoding them.